HPLC Separation of Dihydroxybenzoic Acids on Newcrom B Column

Dihydroxybenzoic acids are aromatic compounds consisting of a phenolic ring and a carboxylic acid. The six main compounds are structurally similar and are difficult to separate in reverse-phase HPLC. The can be separated by using a mixed-mode Newcrom B column with the mobile phase having either methanol (MeOH) or acetonitrile (ACN) as an organic modifier having different retention characteristics. Using the gradient of organic modifier, water and sulfuric acid (H2SO4) as buffer, dihydroxybenzoic acids can be separated and UV detected at 250nm.


Column Newcrom B, 4.6x150 mm, 5 µm, 100A
Mobile Phase MeCN
Buffer H2SO4
Flow Rate 1.0 ml/min
Detection UV, 250 nm


Class of Compounds Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements
Analyzing Compounds 3.4-dihydroxybenzoic acid, 3.5-dihydroxybenzoic acid,2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid
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Application Analytes:
2,3-Dihydroxybenzoic Acid
2,4-Dihydroxybenzoic Acid
2,5-Dihydroxybenzoic Acid
2,6-Dihydroxybenzoic acid
3,4-Dihydroxybenzoic Acid
3,5-Dihydroxybenzoic Acid